Project Summary: Effects of IgE Blockade on T Cells in Food Allergy Food allergies can lead to near-fatal or fatal anaphylaxis. Approximately 30% of food allergic individuals have multiple food allergies. Although many studies have evaluated the efficacy of oral immunotherapy (OIT) for single foods, studies evaluating OIT to multiple foods (multi-OIT) have been limited due to efficacy and safety concerns. Multifood allergic individuals could benefit from treatment, and omalizumab (anti-IgE blockade therapy) potentially mitigates their risk for IgE-mediated allergic reactions. A few groups, including ours, have demonstrated important immunophenotypic and functional biomarker changes induced by single-allergen OIT in T cell subsets. The salient findings from these reports show: increases in the numbers and function of regulatory T cells (Treg); reprogramming of T helper 2 cells (Th2) to the T helper 1 (Th1) subtype; and anergy in allergen-specific Th2 cells. It is of great interest to identify T cell immune biomarker changes while multi-OIT is given to multi-allergic individuals to track desensitization, a lack of clinical reactivity with regular antigen (Ag) exposure, as distinct from sustained unresponsiveness, in which the patient exhibits a long-term and perhaps permanent loss of reactivity to Ag that is independent of continued Ag exposure. Therefore, we have performed a randomized, controlled, phase 2 study in a cohort of multifood allergic participants (Multi Immunotherapy to Test Tolerance and Xolair, ClinicalTrials.gov Identifier: NCT02626611, n=70 participants): peripheral blood mononuclear cells and plasma samples were collected and stored throughout the duration of study. We propose to use these samples and two sets of matched controls for this project. Our main hypothesis is that multi-OIT results in marked downmodulation of Th2 function and concomitant enhancement in Th1 and Treg function, and that these changes will be associated with sustained unresponsiveness and, to a lesser extent, desensitization. To test this hypothesis, we propose to: (Aim 1) Characterize the immunophenotypic and functional changes induced by multi-OIT in total and allergen-specific T cell populations in multi-allergic study participants; (Aim 2) Use MHC class II multimer-based methods to sort allergen-specific single cells, and perform targeted RNA-Seq to investigate their molecular signatures and clonal ancestry at single-cell resolution; and (Aim 3) Quantify epigenetic changes (i.e., methylation of CpG islands) in key genes (i.e., FOXP3, IL4, IFNg, IL10) to assess possible links between the methylation and the desensitization and sustained unresponsiveness resulting from OIT. The results from this study of T cell phenotype, function and epigenetics will enable us: to identify which of these immune features will be most useful as signatures of multi-OIT-induced desensitization and sustained unresponsiveness; to identify patterns of changes in T cells that are associated with these distinct clinical outcomes of multi-OIT; and to determine how these patterns are modified by adding treatment with omalizumab to the multi-OIT protocol.